Preliminary Mechnism Study On Fatty Acid Ester Triggering Corynebacterium Crenatum L-arginine Overproduction

L-arginine(L-Arg),as a kind of semi-essential amino acid,has been widely applied in food additives,pharmaceutical industry and cosmetic.Recently,microbial fermentation methods have been mainly used for L-Arg production.Tween 40,as a fermentation trigger,has been used in L-glutamate overproduction in Corynebacterium glutamicum,and the relative mechanisms also have been illustrated in a certain extent.In this paper,we explored the effect of different kinds of fatty acid ester addition on physiological metabolism and related mechnisims of Corynebacterium crenatum strain-producing L-arginine.Firstly,to investigate the effect of Tween 40 on physiological metabolism of C.crenatum MT NAGK M4 ?proB?Ncgl1221 strain-producing L-arginine,5mg/mL of Tween 40 was added in the early stage of logarithmic growth phase.The result showed that the L-Arg production had a 27.56% increase to 17.14 g/L.Meantime,the intracellular ODHC activity and NADPH concentration were also monitored,and the results found that the ODHC activity was decreased by 20.23% and the NADPH level was enhanced by 131%.Secondly,the dtsR1 gene(encoding the acetyl-CoA carboxylase)and the farR gene(encoding the Fatty acyl-responsive regulator)were knocked out in the C.crenatum CCM01 strain for further increasing the L-Arg production.The results found that the ODHC activity was significantly decreased to 20% and the intracellular NADPH concentration was markedly increased to 0.547 mmol/L by DtsR1 disruption,which could provide the sufficient precursor and reduce power for L-Arg synthesis.L-Arg concentration reached at 27.4 g/L.Moreover,the intracellular NADPH level could be further enhanced by adding different kinds of fatty acid ester in dtsR1 disruption strain,such as Tween 40,Tween 80 and oleic acid.Specially,the conditions of oleic acid emulsified with Tween 80 could be conductive to NADPH accumulation to largest extent,and L-Arg production reached maximum(38.74 g/L).One of possible explanation was that fatty acid ester could induce farR gene down-regulated and indirectly trigger the genes involved in the NADPH synthesis up-regulated.However,in farR disruption strain cultured with oleic acid emulsified with Tween 80,the L-Arg production and NADPH level were kept constantly but the DCW was seriously inhibited.It demonstrated that FarR protein was an essential signal transduction protein in C.crenatum.Finally,in order to investigate the relationship between fatty acid ester and the L-Arg production in C.crenatum mutant strains,the transcription levels of related genes involved in L-Arg biosynthetic network were monitored by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).The results found that the transcription levels of genes involved in encoding and regulating ODHC activity were altered,such as the odhA,sucB,lpdA,pknG,farR and dtsR1 genes were down-regulated,and the ppp gene was up-regulated,and these genes were respectively down/up-regulated to larger extent after DtsR1 disruption.Specially,the zwf gene involved in NADPH synthesis was up-regulated by 5.5 folds in Tween 40 added condition.Interestingly,besides Tween 40 addition,the transcription levels of farR gene were also down-regulated under the Tween 80 or oleic acid emulsified with Tween 80 conditions;and it indirectly induced the gene clusters of tkt-tal-zwf-opcA-pgl,gnd,argB,argG,and gdh genes overexpression.Although these genes were up-regulated in farR knocked-out strain,it could not be conductive to L-Arg overproduction.Therefore,keeping farR low-expression during L-Arg production phase would be a rational strategy for further L-Arg overproduction.